Cardiolipin Supports Respiratory Enzymes in Plants in Different Ways
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fpls-08-00072 February 6, 2017 Time: 17:2 # 1 ORIGINAL RESEARCH published: 08 February 2017 doi: 10.3389/fpls.2017.00072 Cardiolipin Supports Respiratory Enzymes in Plants in Different Ways Jakob Petereit1†, Kenta Katayama2,3†, Christin Lorenz4, Linda Ewert1†, Peter Schertl1, Andreas Kitsche5, Hajime Wada3, Margrit Frentzen6, Hans-Peter Braun1 and Holger Eubel1* 1 Institute of Plant Genetics, Leibniz Universität Hannover, Hannover, Germany, 2 Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan, 3 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan, 4 Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Dortmund, Germany, 5 Institute of Biostatistics, Leibniz Universität Hannover, Hannover, Germany, 6 Institute of Biology I, RWTH Aachen University, Aachen, Germany Edited by: Ruediger Hell, Heidelberg University, Germany In eukaryotes the presence of the dimeric phospholipid cardiolipin (CL) is limited Reviewed by: to the mitochondrial membranes. It resides predominantly in the inner membrane Luning Liu, where it interacts with components of the mitochondrial electron transfer chain. CL University of Liverpool, UK deficiency has previously been shown to affect abundances of the plant NADH- R. George Ratcliffe, University of Oxford, UK dehydrogenase complex and its association with dimeric cyctochrome c reductase. *Correspondence: Using an Arabidopsis thaliana knock-out mutant for the final enzyme of CL biosynthesis Holger Eubel we here extend current knowledge on the dependence of plant respiration on CL. By [email protected] correlating respiratory enzyme abundances with enzymatic capacities in mitochondria †Present address: Jakob Petereit, isolated from wild type, CL deficient and CL complemented heterotrophic cell culture ARC Centre of Excellence in Plant lines a new picture of the participation of CL in plant respiration is emerging. Energy Biology, The University Data indicate a loss of a general reduction of respiratory capacity in CL deficient of Western Australia, Crawley, Australia mitochondria which cannot solely be attributed to decreased abundances or capacities Kenta Katayama, of mitochondrial electron transfer protein complexes and supercomplexes. Instead, RIKEN Center for Sustainable Resource Science, Saitama, it most likely is the result of a loss of the mobile electron carrier cytochrome c. Japan Furthermore, enzymes of the tricarboxylic acid cycle are found to have lower maximum Linda Ewert, activities in the mutant, including the succinate dehydrogenase complex. Interestingly, Institute of Plant Nutrition, Leibniz Universität Hannover, Hannover, abundance of the latter is not altered, indicative of a direct impact of CL deficiency on Germany the enzymatic capacity of this electron transfer chain protein complex. Specialty section: Keywords: plant mitochondria, cardiolipin, protein complexes, protein supercomplexes, respiration, This article was submitted to cytochrome c Plant Physiology, a section of the journal Frontiers in Plant Science INTRODUCTION Received: 02 November 2016 Accepted: 12 January 2017 The dimeric phospholipid cardiolipin (CL) is exclusively found in mitochondria and bacteria. It Published: 08 February 2017 is of highest concentration in the inner mitochondrial membrane and contributes approximately Citation: 10% toward the total lipid content of this organelle (Ardail et al., 1990; Osman et al., Petereit J, Katayama K, Lorenz C, 2011). A single CL molecule comprises four acyl chains connected by a bridge of three Ewert L, Schertl P, Kitsche A, glycerol moieties, themselves interlinked by two phosphate groups. This composition holds Wada H, Frentzen M, Braun H-P and responsible for its wedge-shaped structure in which a bulky fatty acid part is joined to a Eubel H (2017) Cardiolipin Supports relatively small polar headgroup. CL synthesis depends on a set of enzymes also involved Respiratory Enzymes in Plants in Different Ways. in the production of other phospholipid species but employs a unique enzyme for the final Front. Plant Sci. 8:72. step of the pathway, cardiolipin synthase (CLS). This enzyme catalyzes the condensation doi: 10.3389/fpls.2017.00072 of phosphatidylglycerol (PG) and cytidinediphosphate-diacylglycerol (CDP-DAG) in the Frontiers in Plant Science| www.frontiersin.org 1 February 2017| Volume 8| Article 72 fpls-08-00072 February 6, 2017 Time: 17:2 # 2 Petereit et al. Cardiolipin in Plant Respiration mitochondria of animals and plants (Katayama et al., 2004; its individual units were quantified and correlated with protein Chen et al., 2006). Fatty acid composition in CL molecules abundances. Results indicate a low reduction in individual is variable and up to 23 different CL species have recently enzyme capacities which are mostly due to reduced protein been identified across five different plant species (Zhou et al., abundances. An exception to this is the succinate dehydrogenase 2016). Insertion of CL into membrane bilayers induces tensions complex, which does not display an altered abundance despite and membrane bends (Gonzalvez and Gottlieb, 2007). Despite clear reductions in its enzyme capacity. The NADH-producing structural differences phosphatidylethanolamine (PE) and CL enzymes of the citric acid cycle are also severely impaired in have overlapping functions, particularly in mitochondrial fusion respect to their enzymatic capacities. (Joshi et al., 2012). Due to steric limitations, the CL phosphate groups are more accessible promoting the interactions of CL with proteins (Lewis and McElhaney, 2009). RESULTS The mobile electron carrier cytochrome c is an important interaction partner of CL. Studies employing yeast and horse Quantitation of CLS in Arabidopsis Cell heart cytochrome c revealed alterations in protein conformation Suspension Cultures of the Homozygous upon binding to CL-containing lipid vesicles. The resulting increase in cardiolipin peroxidation capacity is accompanied by KO-Mutant Line and Its Corresponding higher levels of membrane pore formation and, consequently, Wild Type Line cytochrome c leakage (Hanske et al., 2012; Bergstrom et al., 2013). Stem tissue of the CLS mutant carrying a T-DNA in the As such, CL: cytochrome c interactions play an important role CLS gene and a complementing gene copy under an estradiol in the early signaling events ultimately leading to the onset of inducible promotor (Katayama and Wada, 2012) was used programmed cell death (PCD) in mammals and yeast (Bergstrom to establish cell cultures providing suitable amounts of plant et al., 2013). Recently, Pineau et al.(2013) and Pan et al.(2014) material for organelle preparations. The site of T-DNA insertion provided strong evidence that CL plays a vital role in protecting is at the start of the fifth exon of the CLS gene (SALK_4984, plants against PCD induced stresses. At4g04870, Katayama and Wada, 2012; Pan et al., 2014). After Besides its role as membrane lipid CL also is a structural 7 days of growth cell mass of the homozygous knock-out component of protein complexes and supercomplexes of the mutant approximately doubled while the WT cell line increased inner mitochondrial membrane such as the ADP/ATP carrier by factor three (data not shown). Complementation of the (AAC, Nury et al., 2005), the bacterial succinate dehydrogenase cls knock-out was induced by adding estradiol to fresh MS- complex (II1, Yankovskaya et al., 2003), as well as the eukaryotic medium to a final concentration of 4 mM prior to subculturing. bc1 complex dimer (III2, Palsdottir et al., 2003), cytochrome c Organelle preparations were performed after 6 or 7 days of oxidase (IV1, Shinzawa-Itoh et al., 2007) and the ATP synthase growth. complex (V1, Eble et al., 1990). Isolated mitochondrial NADH To monitor the success of the T-DNA insertion as well dehydrogenase complex (I1) and III2, rely on CL for restoring the as the complementation strategy in the cell culture system, respective enzymatic activities after phospholipid depletion (Fry targeted selected ion monitoring mass spectrometry (tSIM-MS) and Green, 1981). In a similar fashion, CL positively influences was performed for two selected tryptic peptides of CLS in isolated stability and activity of yeast II1 while simultaneously reducing mitochondria of the knock-out mutant (cls), complemented superoxide anion production of II1 upon reconstitution in CL- mutant (cls/CLS), wild type (WT), and estradiol treated WT containing nanodiscs (Schwall et al., 2012). In addition, CL is cell cultures (WTC). The peptides (LLQSATPLHWR and involved in stabilizing yeast respiratory supercomplexes III2IV1 DLLHPGLVGIVLLR) were chosen for quantitation due to their and III2IV2,(Zhang et al., 2002; Pfeiffer et al., 2003). Their traceability in shotgun MS/MS runs, their uniqueness within reconstitution in lipid vesicles also directly depends on CL (Bazan the Arabidopsis proteome, and the low amount of expected et al., 2013) and other lipids are not or only poorly able to modifications due to the absence of cysteines and methionines. substitute for CL. The mono-isotopic mass and the following two masses of the Since the bulk of CL research has been conducted on yeast isotope distribution of triply charged peptides were taken for mitochondria and bacteria a picture of the dependence of plant this analysis (Figure 1). Results indicate comparable amounts mitochondrial functions on CL is only beginning to emerge. of CLS in WT and WTC but higher average values for In Arabidopsis CL deficiency has recently been linked